Widely dispersed p53 mutation in respiratory epithelium. A novel mechanism for field carcinogenesis

Individuals with one aerodigestive tract malignancy have a high incidence of second primary aerodigestive tumors. The mechanism for this field effect has not been determined. We studied an individual with widespread dysplastic changes in the respiratory epithelium but no overt carcinoma. The entire tracheobronchial tree obtained at autopsy was embedded in paraffin, and bronchial epithelial cells were isolated by microdissection. DNA extracted from the microdissected cells was analyzed for point mutations in the p53 tumor suppressor gene. A single, identical point mutation consisting of a G:C to T:A transversion in codon 245 was identified in bronchial epithelium from 7 of 10 sites in both lungs. Epithelium at sites containing the p53 mutation was morphologically abnormal, exhibiting squamous metaplasia and mild to moderate atypia. No invasive tumor was found in the tracheobronchial tree or any other location. Cells from peripheral blood, kidney, liver, and lymph node exhibited no abnormality in the p53 gene. The widespread presence of a single somatic p53 point mutation in the bronchi of a smoker suggests that a single progenitor bronchial epithelial clone may expand to populate broad areas of the bronchial mucosa-a novel mechanism for field carcinogenesis in the respiratory epithelium that may be of importance in assessing individuals for risk of a second primary tumor as well as in devising effective strategies for chemoprevention of lung cancer.     

Molecular cloning, cDNA sequence analysis, and chromosomal localization of mouse Pkd2

The gene responsible for the second form of autosomal dominant polycystic kidney disease, PKD2, has recently been identified. We now describe the cloning, genomic localization, cDNA sequence, and expression analysis of its murine homologue, Pkd2. The cloned cDNA sequence is 5134 bp long and is predicted to encode a 966-amino-acid integral membrane protein with six membrane-spanning domains and intracellular NH2 and COOH termini. Pkd2 is highly conserved with 91% identity and 98% similarity to polycystin-2 at the amino acid level. Pkd2 mRNA is widely expressed in mouse tissues. Pkd2 maps to mouse Chromosome 5 and is excluded as a candidate gene for previously mapped mouse mutations resulting in a polycystic kidney phenotype.     

Protein kinase C regulation of ATP-induced phosphoinositide hydrolysis in bovine aorta endothelial cells

This study investigated the mechanism of protein kinase C-mediated inhibition of ATP-induced phospholipase C activation in cultured bovine aorta endothelial cells (BAEC). In BAEC labeled with 3H-inositol, phorbol myristate acetate (PMA) prevented ATP-induced inositol bisphosphate and inositol trisphosphate formation. In membranes prepared from these PMA-treated cells, Ca(2+)-, sodium fluoride-, GTP gamma S-, and ATP plus GTP gamma S-stimulated inositol bisphosphate, but not inositol trisphosphate, formation was inhibited. Inositol trisphosphate phosphatase activity was not altered in membranes from PMA-treated BAEC. These results suggest that 1) protein kinase C inhibits ATP-induced phospholipase C activation in BAEC through interference with the coupling of phospholipase C with a G-protein and through an effect on phospholipase C itself, and 2) different mechanisms are responsible for the inhibition by protein kinase C of the phospholipase C-mediated hydrolysis of phosphatidylinositol bisphosphate and phosphatidyl-inositol phosphate.     

Ultrasound diagnosis of metastatic melanoma of the gallbladder

The abdominal ultrasound examinations of 464 patients with malignant melanoma performed over a 3 year period were reviewed. 23 (5.2%) had soft tissue material attached to the gallbladder wall and projecting into the lumen. Four of these were polyps of less than 1 cm which were thought to be benign, while the remaining 19 had abnormalities likely to be metastatic melanoma. Upper abdominal ultrasound examinations are frequently requested for staging purposes in patients with thick high grade malignant melanoma or clinical suspicion of metastases. Ultrasound clearly identifies the gallbladder and biliary tree in the vast majority of patients and is generally regarded as the imaging modality of choice for suspected gallbladder pathology. As autopsy studies have confirmed the incidence of gallbladder metastases from malignant melanoma to be 15-20%, a careful review of the gallbladder is advocated when abdominal ultrasound examinations are performed on patients with malignant melanoma.     

Misinterpretation of body sensations in panic disorder

Cognitive accounts of panic predict that panic disorder patients will be particularly prone to misinterpret autonomic sensations. Several studies have produced results consistent with this prediction, but each is open to alternative interpretation. To clarify matters, 2 studies administered the Body Sensations Interpretation Questionnaire (BSIQ) to panic patients and controls. Panic patients were more likely to interpret ambiguous autonomic sensations as signs of immediately impending physical or mental disaster and were more likely than other anxiety disorder patients and nonpatients to believe these interpretations. In a 3rd study, a brief version of the BSIQ was shown to have satisfactory test-retest reliability, to change with treatment, and to discriminate treatments that varied in their effects on panic.     

Risk factors for Kaposi's sarcoma in HIV-positive subjects in Uganda

BACKGROUND: Kaposi's sarcoma (KS) is associated epidemiologically with HIV infection and with human herpesvirus 8 (HHV-8 or KSHV). Both KS and HIV infection are common in Uganda. We conducted a case-control study of 458 HIV-seropositive. Ugandan adults with KS and 568 HIV-seropositive subjects without KS to examine risk factors for HIV-associated KS. METHODS: We recruited newly diagnosed adult KS cases from five hospitals in Kampala, Uganda and controls from a large referral clinic for HIV infection at Mulago Hospital. All cases and controls were counselled and tested for HIV and answered an interviewer-administered questionnaire about their home, socio-economic conditions, lifestyle and sexual behaviour before they became ill. Only HIV-seropositive subjects were included in the analysis. RESULTS: There were 295 males and 163 females with KS and 227 male and 341 female controls. Age distribution was similar but there was a higher proportion of cases (45%) than controls (29%) residing in rural regions of Uganda. KS cases were more likely than controls to have a higher level of education (X2 for trend, 4.8; P = 0.03), to have occupations associated with affluence [chi 2 for heterogeneity, 17.3 on 5 degrees of freedom (df); P = 0.004] and to come from larger settlements [adjusted odds ratio (OR) for settlements of > 1000 versus 10-99 houses, 1.8; 95% confidence interval (CI), 1.1-3.0]. Cases were more likely than controls to have high household income (chi 2 for trend, 32.6; P < 0.001) and other markers of urban or rural wealth such as owning several cows (chi 2 for trend, 9.5; P = 0.002). Cases were more likely to travel away from home (adjusted OR, 1.6; 95% CI, 1.1-2.3) and more likely to have spent increasing time in contact with water (chi 2 for trend, 12.3; P < 0.001). Few indices of sexual behaviour were related to risk of KS, including reported number of sexual partners. Cases were more likely than controls to be married to one rather than several spouses (adjusted OR, 1.6; 95% CI, 1.2-2.2) and to have reported a history of sexually transmitted diseases (STD) (adjusted OR, 1.6; 95% CI, 1.2-2.3). CONCLUSIONS: Among HIV-infected subjects, KS cases are characterized by better education and greater affluence, compared with controls. Urban address, travel away from home, exposure to water, monogamous marriage and self-reported STD were also more frequent among KS cases than controls. The higher socio-economic status of persons with HIV and KS may be a marker for enhanced exposure to a possibly sexually transmitted agent, or for a delayed exposure to a childhood infection. The risk posed by exposure to water among KS cases requires further study.     

Diverse Fab specific for acetylcholine receptor epitopes from a myasthenia gravis thymus combinatorial library

The muscle weakness in myasthenia gravis (MG) is caused by heterogeneous high-affinity IgG autoantibodies to the nicotinic acetylcholine receptor (AChR), a complex ion channel glycoprotein. These antibodies are clearly responsible for reducing AChR numbers at the neuromuscular junction in myasthenia; however, the origins, diversity, specificity and pathogenicity of individual antibodies have not yet been established. We have cloned and characterized four different AChR-specific Fab from an MG patient's thymus by screening an IgG1/kappa gene combinatorial lambda phage library with soluble human AChR labeled with [125I] alpha-bungarotoxin. Unlike most previously cloned human antibodies, all four Fab immunoprecipitated soluble human muscle AChR. Two Fab strongly inhibited binding of mAb to the main immunogenic region on the alpha subunits and one Fab bound to an epitope on the fetal-specific gamma subunit. In sensitivity and fine specificity, these Fab resembled the anti-AChR antibodies found in many MG patients, including the donor. The closest germline counterparts for their heavy chains were in VH families 1, 3 and 4; however, there were many differences consistent with an antigen-driven response of diverse B cell clones. The combinatorial approach holds promise for further analysis of human autoantibodies.     

A cohort study of pregnancy outcome after amniocentesis in twin pregnancy

We conducted a retrospective cohort study to assess the risk of amniocentesis in twin pregnancy for adverse outcomes. The study base consisted of women who had an amniocentesis performed during twin pregnancy and a comparison representative sample of women who carried a twin pregnancy, but did not have invasive prenatal diagnosis. The 227 women in each of the exposed and non-exposed groups were residents of the state of New South Wales, Australia, over the period 1980-92, and were matched on maternal age and period of the infant's birth. Nearly 10% of twin pregnancies among the women having an amniocentesis were affected by a stillbirth, and the stillbirth rate among exposed fetuses (5.3%) was nearly twice as high as among non-exposed fetuses (3.1%). After adjustment for confounding and excluding abnormalities, there was a non-significant elevated relative risk of stillbirth after exposure to amniocentesis. The analysis by type of amniocentesis (with and without methylene blue dye) was limited by small numbers, but the burden of risk was primarily among women who had dye exposure during amniocentesis (relative risk = 3.64, 95% confidence interval = 1.15, 11.48). This increase remained after adjusting for confounding, although the confidence interval was wide. In conclusion, we were unable to establish with certainty whether an increased risk of stillbirth could be ruled out among women who had any type of amniocentesis in twin pregnancy.     

Dependence of the Ca2+-inhibitable adenylyl cyclase of C6-2B glioma cells on capacitative Ca2+ entry

The ability of adenylyl cyclases to be regulated by physiological transitions in Ca2+ provides a key point for integration of cytosolic Ca2+ concentration ([Ca2+]i) and cAMP signaling. Ca2+-sensitive adenylyl cyclases, whether endogenously or heterologously expressed, require Ca2+ entry for their regulation, rather than Ca2+ release from intracellular stores (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155; Fagan, K., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444). The present study compared the regulation by capacitative Ca2+ entry versus ionophore-mediated Ca2+ entry of an endogenously expressed Ca2+-inhibitable adenylyl cyclase in C6-2B cells. Even in the face of a dramatic [Ca2+]i rise generated by ionophore, Ca2+ entry via capacitative Ca2+ entry channels was solely responsible for the regulation of the adenylyl cyclase. Selective efficacy of BAPTA over equal concentrations of EGTA in blunting the regulation of the cyclase by capacitative Ca2+ entry defined the intimacy between the adenylyl cyclase and the capacitative Ca2+ entry sites. This association could not be impaired by disruption of the cytoskeleton by a variety of strategies. These results not only establish an intimate spatial relationship between an endogenously expressed Ca2+-inhibitable adenylyl cyclase with capacitative Ca2+ entry sites but also provide a physiological role for capacitative Ca2+ entry other than store refilling.     

Polymerase recognition of synthetic oligodeoxyribonucleotides incorporating degenerate pyrimidine and purine bases

A universal base that is capable of substituting for any of the four natural bases in DNA would be of great utility in both mutagenesis and recombinant DNA experiments. This paper describes the properties of oligonucleotides incorporating two degenerate bases, the pyrimidine base 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one and the purine base N6-methoxy-2,6-diaminopurine, designated P and K, respectively. An equimolar mixture of the analogues P and K (called M) acts, in primers, as a universal base. The thermal stability of oligonucleotide duplexes were only slightly reduced when natural bases were replaced by P or K. Templates containing the modified bases were copied by Taq polymerase; P behaved as thymine in 60% of copying events and as cytosine in 40%, whereas K behaved as if it were guanine (13%) or adenine (87%). The dUTPase gene of Caenorhabditis elegans, which we have found to contain three nonidentical homologous repeats, was used as a model system to test the use of these bases in primers for DNA synthesis. A pair of oligodeoxyribonucleotides, each 20 residues long and containing an equimolar mixture of P and K at six positions, primed with high specificity both T7 DNA polymerase in sequencing reactions and Taq polymerase in PCRs; no nonspecific amplification was obtained on genomic DNA of C. elegans. Use of P and K can significantly reduce the complexity of degenerate oligonucleotide mixtures, and when used together, P and K can act as a universal base.